This invention relates generally to a method and apparatus for identifying or screening cells to obtain an absolute count of the cells and/or a multi-part differential/absolute count for research or diagnostic purposes. More particularly, the invention is directed to optically identifying and counting cells which have specific surface molecules, in a known sample volume to obtain a four part differential and/or the absolute cell count of the population of interest, not previously obtainable by simple optical techniques. Stained cells are the basis of conventional cell morphology study. Typically the cells are placed on a slide, and then stained, where they are optically or visually viewed through a microscope. The optical or visual information from microscopic images also can be utilized in automatic scanning devices such as image analyzers.
In the late 19th century, Ehrlich reported on the morphology, physiology and pathology of blood cells, which advanced hematology into a new era by establishing methods of detecting and differentiating the leukemias and anemias. Ehrlich observed that acidic, basic and neutral dyes react specifically with such cellular components as granules and nuclei of white blood cells (WBC).
Romanowsky continued these advancements in hematology by developing a polychromatic stain for use on cells. Currently, Wright's Stain, a modification of Romanowsky's stain is conventionally utilized to visually examine cells. Peripheral blood smears are stained and routinely visually examined for abnormal morphologic variations. Classification of the types of cells and the stages of cell differentiation are key factors in identifying a disease process. Despite the utilization of automated hematology analyzers and flow cytometry instruments, there remains a need for direct visual (microscopic) evaluation of the cells.
Immunologic studies also are important when anomalies are found on a peripheral blood smear. It is necessary to determine the specific subtype of the leukemia in order to better select a treatment method for the disease and to provide the patient with as specific a prognosis as possible. For example, in forms of acute leukemia, there is a predominance of blasts in the peripheral blood. These immature cells can be difficult to classify as either lymphocytic or granulocytic because of the lack of differentiation. If the blast subpopulation that is rapidly proliferating is found to be T11 receptor bearing, the leukemia can be classified as an acute lymphoblastic leukemia, T-cell type. In general, T lineage ALL has a poorer prognosis than B lineage ALL. Further subgrouping these leukemias according to their level of differentiation is also customary. Groups I and II exhibit antigens that are seen on early thymic precursor cells; while those expressed in Group III are similar to the surface antigens found on mature T cells.
Immunology experiments were first developed utilizing a light microscope for determination of lymphocyte subsets. Rosette formation between human lymphocytes and sheep red blood cells (RBC) was observed by Coombs and others in 1970. Later studies found that all or at least a major portion of thymus-derived lymphocytes (T-cells) under the proper conditions displayed the rosette formation phenomenon. These studies utilized Ficoll isolated lymphocytes and were for a period of time routinely employed for subset classification of isolated lymphocytes utilizing a light microscope.
Lymphocyte subsets now conventionally are determined by fluorescent labeling of the cells, in a sample with a fluorescent-tagged monoclonal antibody. The fluorescent-tagged monoclonal antibody binds to the antigen of interest on the surface of the cells expressing the antigen. The cell sample then is analyzed by utilizing a fluorescent microscope or by utilizing a highly sophisticated flow cytometry instrument. When utilizing a flow cytometry instrument, the cell sample preparation, data collection and data analysis must be performed by a specially trained technician. The flow cytometry instrument includes a laser and complex optical system, a high-power computer and electrical and fluidic systems. The component systems of the flow cytometry instrument must be properly maintained and calibrated on a regular and frequent basis. Although the flow cytometry instrument currently is the reference lymphocyte subset determination method, the method has several drawbacks including the high cost of the instrument and the expertise required to correctly operate such instrument.
Lymphocyte subsets also can be determined utilizing automated instruments and methods developed by the assignee of the present application, Coulter Electronics, Inc. An improved simple automated instrument and methods of using the same is disclosed in U.S. application Ser. No. 587,646, filed Sep. 20, 1990, entitled AUTOMATED ANALYZER AND METHOD FOR SCREENING CELLS OR FORMED BODIES FOR ENUMERATION OF POPULATIONS EXPRESSING SELECTED CHARACTERISTICS, which is a continuation of U.S. Ser. No. 025,345, filed Mar. 13, 1987 of the same title. This application combines the application of electronic sensing aperture principles, the specificity of selected biological molecules for identifying and/or enumerating defined populations of cells or formed bodies and microscopic particle technology. The automated analyzer can be used together with a special lysing reagent and/or antibodies coupled to microscopic microspheres or supports of varying composition.
A second application, U.S. Ser. No. 07/849,481, filed Mar. 10, 1992, now issued as U.S. Pat. No. 5,223,398, which is a continuation of U.S. Ser. No. 285,856, filed Dec. 16, 1988, now issued as U.S. Pat. No. 5,231,005, entitled METHOD AND APPARATUS FOR SCREENING CELLS OR FORMED BODIES WITH POPULATIONS EXPRESSING SELECTED CHARACTERISTICS, discloses the screening of direct subsets from whole blood samples or portions thereof.
A third application, U.S. Ser. No. 07/929,156 filed Aug. 12, 1992, now issued as U.S. Pat. No. 5,260,192, which is a continuation of U.S. Ser. No. 339,156, filed Apr. 14, 1989, entitled METHOD AND APPARATUS FOR SCREENING CELLS OR FORMED BODIES WITH POPULATIONS EXPRESSING SELECTED CHARACTERISTICS UTILIZING AT LEAST ONE SENSING PARAMETER, discloses multipart or five part white blood cell differentials, lymphocyte subsets and overlapping determinations performed from a whole blood sample or from a sample with the red blood cells and/or populations of the white blood cells removed by elimination of populations and/or subsets thereof with one or more light or electronic parameters.
A fourth application, U.S. Ser. No. 07/525,231, filed May 17, 1990, entitled METHOD AND APPARATUS FOR SCREENING OBSCURED OR PARTIALLY OBSCURED CELLS, discloses an analysis of obscured cells by utilizing microspheres having specific monoclonal antibodies bound thereto to move the sensed characteristics of the obscured cells from one cell population to another. Each of the above four referenced applications is incorporated herein by reference.
The method and apparatus embodying the invention can be utilized with a variety of immunological reactions, such as immunological reactions involving reactants and formed bodies or cells. The invention also applies to analyses of formed body suspensions such as some bacteria and viruses among others. As utilized herein, cells are defined as animal or plant cells, including cellular bacteria, fungi, which are identifiable separately or in aggregates. Cells are the least structural aggregate of living matter capable of functioning as an independent unit. For example, cells can be human red blood cell (RBC) and WBC populations, cancer or other abnormal cells from tissue or from blood samples. Formed bodies are defined as some bacteria and viruses. The cells and formed bodies suitably tagged or labeled, reasonably can be expected to be optically identified by the method and apparatus of the invention in the same manner as the human blood cell examples.
Although the term "reactant" has been utilized in the above applications to define lysing agents and monoclonal antibodies, reactants can include various agents which detect and react with one or more specific molecules which are on the surface of a cell or formed body. Some examples are given below:
______________________________________ Reactant Specific Molecule ______________________________________ Antibody Antigen Drug Drug Receptor Hormone Hormone Receptor Growth Factor Growth Factor Receptor ______________________________________
The reactants couple or bind to the specific molecule(s) on the cells. These reactants do form part of a chemical reaction; however, the reactants are not necessarily chemically altered.
One prior art procedure of lymphocyte subset determination utilizes a light microscope and antibody-labeled microspheres. This procedure is available from Bio-Rad Laboratories of Richmond, Calif. The procedure is available to identify T and B lymphocytes. The antibody-labeled microspheres are utilized to bind to the cells which exhibit the surface antigen of interest. Two different colored antibody-labeled microspheres are utilized to differentiate the T and B lymphocytes. The microscopist identifies the cells positive for a particular antigen by the presence of the antibody-labeled microspheres bound to the cells. Cells which do not have an antibody-labeled microsphere bound thereto represent the negative cell population, not expressing the antigen(s) of interest. This procedure is limited by the fact that the lymphocytes must first be isolated from a whole blood sample, utilizing an isolation medium, such as Ficoll-Hypaque. Granulocyte contamination can lead to falsely elevated non-positive cell values and an increased number of phagocytic cells. Further, only lymphocyte subset determinations can be performed, since the remaining cells from a whole blood sample first are eliminated. Also, the cell morphologic evaluation is limited since the cells are not stained with the conventional Romanowsky or Wright type stain, but merely are stained to determine the cell viability. The morphologic evaluation is further limited since the cells are evaluated in a suspension and are not evaluated on slides, as is conventional.
An improved method and apparatus for optically screening cells to identify the morphology and selected characteristics or properties expressed by the cells is disclosed in parent application U.S. Ser. No. 617,096, filed Nov. 23, 1990 entitled METHOD AND APPARATUS FOR OPTICALLY SCREENING MICROSCOPIC CELLS, incorporated herein by reference. The cells are combined with one or a plurality of different sets of microspheres, each set having a reactant bound thereto which will bind to a specific molecule which can exist on one or more types of the cells. The cells and microspheres are prepared as a smear on a slide and stained with a Wright type stain. The cells then are optically viewed by an operator and/or automatically, to identify the type of cells, if any, to which the different microspheres are bound.